Given the high rate of sexual transmission of HIV-1, particularly in the developing world, the need for a topical microbicide is critical. The long term goal of this research is to develop an anti-HIV microbicide using HIV-1 fusion inhibitors. In particular, we have found that the combination of certain chemokine variants with gp41-binding proteins results in highly potent inhibition of HIV-1, both in fusion assays (IC50= 1 pM) and in viral assays in PBMC (IC50=0.7 nM, R5 tropic strain Ba-L). The Aims of the proposal are as follows: First, combinations of CCR5 binding proteins and gp41-binding peptides will be tested in both fusion assays and in viral assays with multiple clades of HIV as well as primary strains in order to determine which combinations provide the most potent protection. Then it will be determined if a higher level of inhibition efficiency can be obtained by combining both a variant chemokine and a gp41-binding protein on a single polypeptide chain. During the R33 phase of the project, it is proposed to carry out pre-clinical evaluation of the most potent inhibitors and combinations, including, stability to pH and ionic strength, cell toxicity and irritation in animal models. The most promising inhibitors will then be tested in two different ways. In Aim 4 they will be expressed by Lactobacillus jensenii, an organism that is naturally found in vaginal mucosa, and as such represents a method of delivery of protein microbicides having great potential. Finally, in Aim 5, the best entry inhibitors will be evaluated in a humanized mouse model that has been shown to be able to be infected with HIV. Anti-HIV microbicides are molecules that can be used topically to prevent the spread of the HIV-1 virus through sexual transmission. The proposed experiments will study the combination of CCR5-binding proteins and gp41-binding proteins to synergistically inhibit HIV and as components of a microbicide.